Not Applicable
This invention pertains to compositions capable of regulating protein activity, particularly activity dependent upon binding between two or more proteins. Specifically, the invention pertains to compositions and methods for delivering a peptide inhibitor moiety into a living cell including moieties, capable of inhibiting binding between a protein kinase and at least one other protein in the cell such as anchoring proteins.
Signaling molecules, such as hormones and neurotransmitters, elicit cellular responses through interrelated biochemical reactions. A variety of specific and selective reagents have been developed, which make it possible to study how such signaling molecules mediate such cellular responses in vitro. The study of such responses is an important first step in the discovery and development of new drugs. The development of specific and selective reagents capable of being used, in similar studies in either cell culture medium or in a whole animal is of equal importance. The most preferred such reagents are, traditionally, ones which enable the study of specific processes in living cells without breaking the cell or introducing artifacts therein.
Synthetic molecules such as synthetic peptides or peptidomimetics capable of interacting with, or competing with signaling protein molecules, such as protein kinases or the insulin receptor, have been reported in the literature. Various strategies have been reported for introducing such synthetic molecules into living cells, including electroporation, osmotic shock, and permeabilization of cells using agents such as saponins, streptolysin O, or liposomes. However, those delivery systems tend to cause cellular damage. Microinjection, another known method for introducing such synthetic molecules into living cells, is less damaging, but suffers the drawback of being labor intensive.
A recent publication by Liotta et al. indicates that synthetic peptides can be modified so that they passively migrate into the membranes of living cells, where the modified peptides can interact with trans-membrane proteins (Liotta et al., J. Biol. Chem. 269:22996-23001, 1994). The Liotta et al. publication demonstrates that a synthetic Tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain is a good inhibitor of tyrosine dephosphorylation of the insulin receptor in situ. The most significant observation made by Liotta et al. was the fact that a particular synthetic tris-sulfotyrosyl dodecapeptide (TRDIYETDYYRK-amide) (SEQ ID NO:1), a stearyl peptide amide, caused an 4.5-fold increase in insulin-stimulated receptor autophosphorylation in intact CHO/HIRc cells. That stearyl peptide amide displayed specificity toward tyrosine-class phosphatases only, as it did not have an effect on the activities of the serine/threonine phosphatases PP-1, PP-2A, or alkaline phosphatase. Approaches similar to that of Liotta et al. but with fatty acid-peptide conjugates had been used to inhibit protein kinase C (PKC) and tyrosine kinase activities in intact cells (Eichholtz et al. J. Biol. Chem., 268: 1982-1986, 1993; Liotta et al., J. Biol. Chem. 269(37):22996-23001, 1994; and O""Brian et al., Biochem. Pharmacol. 39:49-57, 1990).
One type of signaling protein molecule, signal transduction enzymes (e.g. protein kinases and phosphatases), play pivotal roles in mediating cellular responses to a wide variety of stimuli. Such enzymes are often targeted to specific substrates or cellular compartments through their interaction with cellular xe2x80x9canchoring proteinsxe2x80x9d (Hubbard and Cohen, Trends Biochem. Sci. 18:172-177, 1993). The anchoring or compartmentalization of such proteins is thought to be critical in determining the specificity of response for a particular stimulus (Scott and Carr, News Physiol. Sci. 7:143-148, 1992; Rubin, Biochem. Biophys. Acta 1224:467-479, 1994; Mochly-Rosen, Science 268:247-251, 1995). Anchoring of cyclic AMP-dependent protein kinase A (PKA or A-kinase) is accomplished by the binding of its regulatory subunit (R) to an amphipathic helix-binding motif located within A-kinase anchoring proteins (AKAPs) (Carr et al., J. Biol. Chem. 266:14188-14192, 1991).
Many different peptides have been developed and synthesized which are known to have functional activities in vitro, including peptides designed to interact with anchor proteins or other proteins known to have binding regions in the intracellular space of living cells. The protein kinase anchoring inhibitory peptides, such as Ht31, (DLIEEAASRIVDAVIEQVKAAGAY) (SEQ ID NO:2), are included in the category of peptides designed to interact with anchor proteins (Carr et al., J. Biol. Chem. 267(19):13376, 1992). Ht31 has been shown to inhibit the binding of the regulatory subunit of PKA to anchoring protein using cellular extracts in vitro. The PKA regulatory subunit from which the sequence of Ht31 was derived was used to synthesize the peptide moieties of the new reagents used in several of the examples of this application. Peptides such as Ht31, which are known to inhibit PKA binding to anchor proteins, are referred to herein as xe2x80x9canchor inhibiting peptidesxe2x80x9d or (AIPs).
Peptides known to be functionally active in vitro have also been used to develop and synthesize inactive peptides for use as control peptides. The alterations made in the amino acid sequence of an active peptide to produce an inactive peptide are expected to have a disruptive effect on the predicted xcex1-helix conformation of the active parent peptide, a conformation considered essential to ensure the maintenance of functional activity. Altered inactive peptides of this type have been used as controls in assaying the functional activity and other properties of the parent peptides from which they were derived. The sequence of any such control peptide is derived from the sequence of its functionally active counterpart by substituting a few amino acid residues in the parent amino acid sequence with other amino acid residues likely to produce a significantly different conformation (See, e.g. Carr et al., supra; and Rosenmund et al., Nature 368:853-856, 1994).
Microinjection of AIPs into neurons or skeletal muscle cells has been shown to disrupt PKA anchoring and PKA modulation of glutamate receptor channels (Rosenmund et al., supra) and voltage-gated calcium channels (Johnson et al., Proc. Natl. Acad. Sci. USA 91:11492-11496, 1994). However, microinjection is impractical for normal pharmaceutical applications. If a more practical method were found for introducing AIPs and other functionally active peptides intracellularly, it would lead to many possible applications, particularly those applications involving the control and regulation of cyclic adenosine monophosphate (cAMP) mediated responses in living cells.
Cyclic AMP (cAMP) is known to mediate the motility of sperm and a variety of other ciliated cells (Satir, Modern Cell Biol. 4:1-46, 1985; Tash, Cell Motil. Cytoskel. 14:332-339, 1989; Bedford and Hoskins, in Marshall""s Physiology of Reproduction, Lamming, ed., pp. 379, Churchill Livingstone, N.Y. 1990). Increases in the level of this nucleotide are associated with development of motility in the epididymis (Bedford and Hoskins, supra; Hoskins et al., J. Reprod. Fertil. 37:131-133, 1974). Cell-permeant cAMP analogs, e.g., cAMP phosphodiesterase inhibitors, and adenyl cyclase activators, all stimulate motility of sperm from several species (Garbers et al., Biol. Reprod. 7:132, 1972; Garbers et al., Adv. Cyclic Nucleotide Res. 9:583-595, 1978; Hoskins, Journal of Biological Chemistry 248:1135-1140, 1973; Hoskins et al., Biol. Reprod. 13:168-176, 1975; Vijayaraghavan and Hoskins, J. Cyclic Nucleotide Protein Phosphoryl. Res. 10:499-510, 1985). The kinetic and metabolic responses to cAMP elevation have been discovered to occur within 5 to 10 minutes (Garbers et al., Biol. Reprod. 7:132, 1972; Garbers et al., Adv. Cyclic Nucleotide Res. 9:583-595, 1978).
Sperm are known to lack nucleic-acid and protein-synthetic activity, thereby considerably reducing the possible range of targets of cAMP action. Sperm have been found to have distinct subcellular structures easily distinguished with the aid of a light microscope. Immunogold staining of sperm, followed by examination under a light microscope, has been used to demonstrate a predominant localization of the type II subunit (RII) of a PKA anchoring protein at the outer membrane of the mitochondria that spiral around the proximal flagella (Lieberman et al., J. Cell Biol. 107:1809-1816, 1988). A developmentally regulated sperm AKAP (i.e., AKAP-84) has also been localized to sperm mitochondria (Lin et al., J. Biol. Chem. 270:27804-27811, 1995).
Cyclic AMP and the proteins which regulate the production of cAMP in living cells, such as the various protein kinase regulatory anchoring proteins (e.g. AKAPs), are important in mediating many different responses in a wide variety of cells, other than the motility response in sperm cells. For example, AKAPs have been found to play a critical role in the regulation of synaptic function in cultured hippocampal neurons (Rosenmund et al., supra).
Therefore, what is needed is a product or process for introducing peptides known to be functionally active into the intracellular space of living cells. As used herein, the term xe2x80x9cintracellular spacexe2x80x9d refers to the region of a cell bounded by the cell membrane or cell wall. In the case of a eukaryotic cell, the term intracellular space refers to everything contained within the cytoplasm of the cell, including all organelles and regions of proteins protruding into the cytoplasm from the inner surface of the cell membrane. Particularly needed is a product or process for introducing AIPs into the intracellular space of living cells and, more particularly, AIPs capable of inhibiting the binding of specific protein kinases to specific protein kinase anchor proteins. Disclosed herein is such a product, e.g., an alkyl peptide amide, and a process for the introduction of at least the amide portion of the product into the intracellular space of living cells.
Also disclosed herein are examples of the synthesis and use of such alkyl peptide amides to inhibit the activity of protein kinase A in HeLa cells and to inhibit binding between the RII subunit of protein kinase A and the anchor binding protein in sperm from various different organisms.
The principal aspects of the invention are as follows. In one aspect the present invention is an alkyl peptide, comprising:
an alkyl moiety having at least twelve (12) carbon atoms;
a peptide moiety, the peptide moiety having an inhibitor region comprising a sequence of amino acids substantially homologous to a binding domain within a first protein, wherein the binding domain is capable of binding a second protein in the intracellular space of a living cell; and
a linkage between the alkyl moiety and the peptide moiety.
In another aspect, the present invention is an alkyl peptide amide, comprising:
an alkyl moiety having at least twelve (12) carbon atoms, the alkyl moiety including a carbonyl terminus;
a peptide moiety, the peptide moiety having an inhibitor region and an N-terminus, the inhibitor region comprising a sequence of amino acids substantially homologous to a binding domain within a first protein, wherein the binding domain is capable of binding a second protein in the intracellular space of a living cell; and
an amide linkage between the carbonyl terminus of the alkyl moiety, and the N-terminus of the peptide moiety.
In a further aspect, the present invention is a pharmaceutical composition comprising an alkyl peptide amide described above in a pharmaceutically acceptable carrier.
In yet a further aspect, the present invention is a method of using an alkyl peptide amide to inhibit the binding of a first protein to a second protein in a living cell, comprising the steps of:
(a) selecting a living cell having a first protein, a second protein, and a cell membrane surrounding an intracellular space, wherein the first protein has at least one binding domain capable of binding the second protein in the intracellular space;
(b) providing an alkyl peptide amide, comprising:
an alkyl moiety of at least twelve (12) carbon atoms, the alkyl moiety having a carbonyl terminus;
a peptide moiety, the peptide moiety having an inhibitor region and an N-terminus, the inhibitor region comprising a sequence of amino acids substantially homologous to the binding domain within the first protein for the second protein; and
an amide linkage between the carbonyl terminus of the alkyl moiety, and the N-terminus of the peptide moiety; and
(c) exposing the alkyl peptide amide to the living cell so that the peptide moiety permeates the cell membrane and inhibits the binding of the first protein to the second protein.
The alkyl peptide amides of this invention are efficient delivery systems for introducing peptide moieties into the intracellular space of living cells, including sperm cells and HeLa cells. The motility of sperm cells from a wide variety of different species can be inhibited after such cells are exposed to the alkyl peptide amide form of AIPs (i.e. alkyl AIP amides). This last form of the alkyl peptide amides of this invention (alkyl AIP amides) can be included in pharmaceutical compositions and used as contraceptives, either alone or in combination with other contraceptive substances, e.g., a spermicidal agent such as nonoxynol-9.
The pharmaceutical compositions of the alkyl peptide amides of this invention include pharmaceutical contraceptive compositions, particularly such compositions comprising an alkyl peptide amide known to inhibit sperm motility, such an alkyl AIP amide. The pharmaceutical contraceptive compositions of this invention can be used in the form of a topical ointment or lotion e.g., to lubricate the vagina before intercourse. Other modes of application of the pharmaceutical composition of the invention are likely to occur to one skilled in the contraceptive art. By inhibiting sperm motility, the contraceptive compositions of this invention could prevent fertilization of the ovum (alone or in combination with other contraception techniques), with minimal side effects.
The pharmaceutical contraceptive compositions of this invention are likely to produce few side effects because: (1) the peptide moiety of the alkyl peptide amide has a substantially similar sequence to a natural cellular component; (2) only very low concentrations of the alkyl peptide amide component are needed for a composition to be effective as an inhibitor of sperm motility; and (3) the effect, i.e., sperm motility inhibition, has been found to be temporary, i.e., to be reversible once sperm cells are no longer in contact with a composition containing the alkyl peptide amide.
The detailed disclosure below demonstrates that the compositions and methods of the present invention can be used to introduce inhibitor moieties, such as peptide or peptidomimetic moieties into the intracellular space of a variety of different types of cells, including sperm cells obtained from various different species, and HeLa cells (a continuously cultured cell line, originally obtained from human cancerous cervical tissue.) Aspects of this invention may also have utility in other applications which will occur to one skilled in this art in light of the disclosure herein. One such aspect is the inhibition of transformation in certain tumorous cells. All such other applications are included within the scope of this invention.